title: Use of an anti-apoptotic CHO cell line for transient gene expression.
abstract: Transient gene expression in mammalian cells allows for rapid production of recombinant proteins for research and preclinical studies. Here, we describe the development of a polyethylenimine (PEI) transient transfection system using an anti-apoptotic host cell line. The host cell line, referred to as the Double Knockout (DKO), was generated by deleting two pro-apoptotic factors, Bax and Bak, in a CHO-K1 cell line using zinc finger nuclease mediated gene disruption. Optimized DNA and PEI volumes for DKO transfections were 50% and 30% lower than CHO-K1, respectively. During transfection DKO cells produced relatively high levels of lactate, but this was mitigated by a temperature shift to 31°C which further enhanced productivity. DKO cells expressed ∼3- to 4-fold higher antibody titers than CHO-K1 cells. As evidence of their anti-apoptotic properties post-transfection, DKO cells maintained higher viability and had reduced levels of active caspase-3 compared to CHO-K1 cells. Nuclear plasmid DNA copy numbers and message levels were significantly elevated in DKO cells. Although DNA uptake levels, as early as 40 min post-transfection, were higher in DKO cells this was not due to differences in cell surface heparan sulfate (HS) or initial endocytosis mechanism as both cell types utilized caveolae- and clathrin-mediated endocytosis to internalize DNA:PEI complexes. These results suggest that the increased transfection efficiency and titers from DKO cells are attributed to their resistance to transfection-induced apoptosis and not differences in endocytosis mechanism.
authors.affiliation: Dept. of Early Stage Cell Culture, Genentech Inc., South San Francisco, CA 94080, USA.
authors.forename: Nichole F
authors.initials: NF
authors.lastname: Macaraeg
authors.forename: Dorothea E
authors.initials: DE
authors.lastname: Reilly
authors.forename: Athena W
authors.initials: AW
authors.lastname: Wong
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000235:genetics*; D016466:CHO Cells; D002478:Cells, Cultured; D003412:Cricetulus; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D015870:Gene Expression / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D012333:RNA, Messenger; D004247:DNA
keywords: Bak; Bax; apoptosis; endocytosis; transfection efficiency; transient gene expression